Ehrlichia spp. are obligately intracellular gram-negative bacteria that reside in endosomes within hematopoietic cells and infect various hosts including humans, domestic and wild canidae, deer, horses, sheep, cattle, and wild rodents. Each member of the family Anaplasmataceae has its own particular primary target cell tropism. Most species of Ehrlichia are either monocytotropic (e.g. E. canis, E. chaffeensis, and E. muris) or granulocytotropic (e.g. Anaplasma phagocytophilum, and E. ewingii) with the exceptions of Ehrlichia ruminantium, which grows in the endothelial cells of the host, Anaplasma marginale, a red blood cell parasite, and Anaplasma platys, a platelet parasite.
Although Ehrlichiae were described in the early part of the 20th century, they received very little attention because they were considered pathogens of only veterinary importance in the United States until the recent two decades. The renewed interest in Ehrlichia is due to the emergence of ehrlichioses affecting humans. Since 1987, three emerging human ehrlichial pathogens (E. chaffeensis, E. ewingii, and A. phagocytophilum) have been discovered in the United States.
Ehrlichia canis, the prototype species of the genus, is the etiologic agent of canine monocytotropic ehrlichiosis (CME). CME, also known as canine tropical pancytopenia, is a worldwide disease transmitted by the brown dog tick, Rhipicephalus sanguineus. The progression of canine ehrlichiosis occurs in three phases: acute, sub-clinical and chronic. The acute phase is characterized by fever, anorexia, depression, lymphadenopathy and mild thrombocytopenia. Dogs typically recover from the acute phase, but become persistently infected carriers of the organism without clinical signs of disease for months or even years. A chronic phase develops in some cases that is characterized by thrombocytopenia, hyperglobulinemia, anorexia, emaciation, and hemorrhage, particularly epistaxis, followed by death. Each year it costs millions of dollars worldwide for treating companion and working dogs infected with E. canis. Ehrlichia canis was isolated from a human recently, and thus may be an emerging public health threat.
One aspect of E. canis biology concerns the need for iron, which is a necessity for survival of nearly all prokaryotes and eukaryotes. For the obligate intracellular bacterium Ehrlichia chaffeensis, the importance of iron for intracellular survival has been demonstrated by inhibition of Ehrlichia proliferation in the presence of the intracytoplasmic iron chelator deferoxamine (Barnewall et al., 1997). Early endosomes containing the bacteria (morulae) upregulate and accumulate the mammalian transferrin receptor (Barnewall et al., 1997), suggesting that the organism has developed specific strategies for iron acquisition. The cytoplasmic vacuole where the Ehrlichia resides is acidic, which may promote the release of free iron into the compartment (Barnewall et al., 1997). An E. chaffeensis protein with homology to known ferric binding proteins has been previously reported (Yu et al., 1999), but the mechanisms of iron acquisition by Ehrlichia spp. are unknown and functional demonstration of iron binding properties of Ehrlichia Fbps has not been experimentally demonstrated. Furthermore, the E. chaffeensis Fbp polypeptide is not immunoreactive.
Iron is involved in many key metabolic functions of the cell, but insoluble Fe(OH)3 (rust) forms at a physiological pH and thus must be stored and transferred via iron binding proteins (Andrews et al., 2003; Mietzner et al., 1998). In eukaryotic systems, extracellular iron-binding proteins include transferrin and lactoferrin, while ferritin binds and sequesters intracellular iron. Protein-bound iron also inhibits the interaction of Fe(II) with O2, preventing the formation of free radicals that are damaging to cells (Andrews et al., 2003; Mietzner et al., 1998). The availability of iron has been demonstrated to increase the virulence of many diverse pathogenic bacteria (Raymond et al., 2003). Limiting the availability of free iron is a mechanism to naturally suppress growth of bacteria. However, under the selective pressure of the limited iron available in the host, pathogenic bacteria have evolved specific iron acquisition mechanisms including iron binding molecules and proteins.
Bacteria commonly utilize siderophores as means of iron mobilization. These molecules are nonproteinaceous iron chelators that are expressed, secreted, and bound by surface receptors that enable the transport of free iron from the environment (Andrews et al., 2003; Mietzner et al., 1998). Another means of iron uptake involves iron acquisition from the host iron binding proteins transferrin and lactoferrin (Andrews et al., 2003; Mietzner et al., 1998). This mechanism, used by many gram-negative pathogens involves a conserved system by which iron is competed away from transferrin or lactoferrin at the outer membrane of the bacterium and the iron is shuttled across the membrane and into the periplasm (Andrews et al., 2003). Mobilization of iron into the cytoplasm involves three proteins that belong to the ATP-binding cassette (ABC) transporter family, the ferric binding protein (Fbp), a cytoplasmic permease, and an ATP-binding protein (Adhikari et al., 1996; Adhikari et al., 1995; Andrews et al., 2003; Clarke et al., 2001; Mietzner et al., 1998), all encoded by an operon system that is shared among many diverse bacterial species including Neisseria gonorrhoeae, Haemophilus influenzae, Mannheimia (Pasteurella) haemolytica, Serratia marcescens, Salmonella typhimurium, and Yersinia pestis (Adhikari et al., 1996; Adhikari et al., 1995 Angerer et al., 1990; Bearden and Perry, 1999; Bearden et al., 1998; Janakiraman and Slauch, 2000; Kirby et al., 1998). The operon system of Neisseria gonorrhoeae has been demonstrated to be under the control of the iron regulatory element Fur (ferric uptake regulator)(Desai et al., 1996).
Identifying the genetic and antigenic compositions of E. canis is essential for studying the pathogenesis of canine ehrlichiosis and developing an effective vaccine. Although Yu et al. (1999) describe an E. chaffeensis 37-kDa protein that is homologous to the iron (III)-binding periplasmic protein precursor of gram-negative bacteria, the prior art is deficient in the cloning and characterization of immunoreactive gene of Ehrlichia canis. The present invention fulfills this long-standing need and desire in the art by cloning an immunoreactive Fbp of Ehrlichia canis. 